The Facts

In dicotyledonous plants the stem apex is a terminal bud. This bud normally produces auxin, mainly from the young developing leaves in it (12,17,331; see also 279), but also to some extent from the stem apex itself (101), and this auxin promotes the development of the stem imme-diately below it. However, the same auxin also prevents the develop-ment of lateral buds lower down on the stem, thus allowing the terminal bud to retain its " apical dominance." When the terminal bud is removed, as in pruning, one or more lateral buds (usually those in the most apical axils remaining) begin to develop ; in so doing they also begin to produce auxin, which in turn inhibits the buds still lower down. If, after removal of the terminal bud, auxin is applied in its place, the lateral buds remain inhibited.

The first demonstration that bud inhibition is due to a diffusible sub-stance was made by Snow (278), who showed that the inhibiting influence coming from the terminal bud in Vicia faba could cross a dis-continuity of tissue; this experiment corresponds with those of Boysen-Jensen and Paâl for the promotion of growth (see Section I). Eight years later Thimann and Skoog confirmed Snow's finding and identified the inhibiting influence with auxin, which at first (330,331) was obtained from Rhizopus, and later (273) with pure indoleacetic acid and auxin b.

Laibach also showed that an inhibiting substance diffused from pollen (180). Confirmation with numerous different plants soon followed (73,81,101,136,337). The concentrations needed for inhibition, though somewhat higher than for growth, are entirely physiological and not toxic, for lateral buds which have been inhibited in this way resume their growth when the auxin source is removed (331). Several different natural and synthetic auxins have been shown to be effective (117,136,

176,273,311). It should be mentioned that leaves also exert an inhibi-tion, though to a lesser extent than the terminal bud, as was shown earlier by Dostâl (80).

2. Mechanisms

The way in which the inhibition is brought about is far from clear.

The many hypotheses have been reviewed by Snow (283) and Thimann (316). At first it was thought that the auxin at the apex (either produced naturally by the bud, or applied artificially after decapitation) in some

way diverted to itself substances necessary for bud growth and thus starved the lateral buds (211,354; see also the discussion in Section V of the following chapter). This is similar to the view of Goebel and other older botanists who considered that a growing apical bud maintained its dominance by using up the available nutrients. A modification of this view is that of Ferman (85), who suggested that the growing bud draws to itself the supply of auxin precursor, so that the laterals are unable to produce auxin. This is supported by the undoubted fact that inhibited buds produce less auxin than do growing buds (331) and, though the evi-dence is not quite consistent, they also appear to contain less total extractable auxin than growing buds (85,228). In other words, there is some reason to think that the inhibition is exerted not so much on the growth of the bud as on its ability to produce auxin.

However, it now seems clear that inhibition cannot be primarily an indirect effect due to the diversion of materials away from the bud, since application of auxin directly to the lateral buds, either in situ on the stem, as in the experiments of Plch (241) and Thimann (314), or isolated and growing in nutrient solution, as by Skoog (272), causes clear-cut inhibi-tion of their growth. Also in small fragments of plant tissue in culture, particularly root tissues, auxins such as naphthaleneacetic acid strongly inhibit the development of buds (96,98). In slices of potato tuber the local application of auxin inhibits bud development without producing any corresponding growth elsewhere (81,202a). Exposure of whole potatoes to auxins in vapor form (i.e., methyl esters of the acids) causes inhibition of all the buds (123). In none of these cases would it seem that the effect can rest on movement of materials elsewhere ; the effect is primarily local.

It appears that the influence of auxin on different organs is represented by a series of optimum curves, intermediate concentrations promoting growth and higher concentrations inhibiting it (45,314), as shown in Fig. 7 (p. 45). Thus the concentrations causing stem growth would be high enough to inhibit bud development. This general theory receives support from the numerous effects of auxin in Gautheret's cultures of various organs (96; see Section VIII, A), and additional considerations which may help to explain it have been advanced by Skoog et al. (272a) ; against it is the lack of any cambial activity in inhibited buds (280) although auxin is known to stimulate the cambium (see Section VIII).

A peculiar and unexplained fact is recorded by Castan (57), namely, that, if high auxin concentration is applied to the intact terminal bud, it loses its power of inhibiting the lateral buds below it.

The problem is made more complicated by the direction in which inhibition is exerted. Since auxin moves polarly from apex to base,

inhibition should be only exerted on buds morphologically below, i.e., basal to, the source of auxin. Although this in general is strictly the case, there are exceptions in which buds are inhibited above the point of auxin application (283,285), and a parallel has been suggested with certain upward inhibitions of stem elongation, studied by Pohl (243), Le Fanu (190), and Mitchell and Martin (204). The phenomena of geotropism, however, provide clear agreement with expectation; here the auxin is known to be diverted to the lower side of stems by gravity, so that we should expect to find that in horizontal stems the buds on the lower side are inhibited; this was observed as long ago as 1917 by Loeb and has been confirmed in different plants by many workers (73,237,249,272).

One type of phenomenon which might have significance for bud inhibition has not yet been brought into the picture. Many workers have found evidence for growth-inhibiting material in plant tissues, par-ticularly in ether extracts thereof. Köckemann (154,155) extracted such material from fruits, demonstrating its effect by inhibiting the germination of seeds. This so-called "blastokolin" was investigated by Kuhn et al. (177), who extracted an oil from Sorbus fruits which strongly inhibited seed germination, and demonstrated that parasorbic acid had similar effects. Other substances having an unsaturated lactone struc-ture (340), including coumarin, act in the same way. Moreover, Voss (342) extracted from corn, and Larsen (186) from tomatoes, material which inhibits growth of the Avena coleoptile. Linser (199) made similar extracts from lilac leaves and showed that they also inhibit the formation of roots. Juel (152), in an extension of Larsen's work, utilized his assay method of mixing the inhibiting extract with known concentrations of auxin in agar and using the Avena test on the mixture.

She showed that the inhibition is not due to auxin inactivation, and that it is exerted also on root growth, which itself is inhibited by auxin (see pp. 43-46). Hence the extracted material is not simply an antiauxin, but an inhibitor of the growth of both shoots and roots. Similar experiments have been carried out with sugar cane nodes, from which the inhibitor is liberated by hot water (237,237a). The dormancy of potato buds has been shown by Hemberg (131a) to be due to an inhibitor present in large amounts in the periderm, and disappearing slowly as the tubers mature.

The auxin content does not change during dormancy but increases shortly before sprouting.

More suggestive still is the inhibitor of Stewart et al. (297,299), which produces a marked positive curvature (i.e., toward the block) in the Avena test. This substance was partially purified and shown to yield an auxin—most probably indoleacetic acid—on alkaline hydrolysis. If it could be shown that lateral buds have the property of producing this

inhibitor directly from auxin, a mechanism of bud inhibition would be at hand. Although as yet there is no such direct evidence, the scheme advanced by Skoog et al. (272a) gives a very plausible rationale for this.

Furthermore, Snow (286) has brought forward independent evidence that bud inhibition is due to a special inhibiting hormone in some way produced by auxin. This concept has recently been discussed by Skoog (274a).

The situation can be summed up by saying that most of the data point to bud inhibition as due to auxin directly, with the mechanism probably involving the formation of an inhibitor by or under the influ-ence of auxin. The possibility is not excluded, however, that other substances necessary for growth may in some way play a part.

3. General Significance

The inhibition of one bud by another is a phenomenon of very wide occurrence and has a broad influence on general morphology. In tubers, for instance, development of a bud at the apical end leads to inhibition of others, but ringing, or physical isolation of these buds, allows the lower buds to develop (81,131,202a). The auxin is presumably carried from one bud to another through the cortex. Auxin application, either as paste to the outer cortex or as vapor to the whole tuber, maintains the buds in the inhibited state, and this is now being used on a large scale in the storage of potatoes, with methylnaphthalene acetate. It is of interest that such auxin-inhibited buds resemble normal dormant buds in that they are stimulated to sprout by ethylene chlorhydrin (123,202a).

This treatment greatly increases the rate of auxin destruction, thus releasing the buds from inhibition; when growth begins again the terminal bud soon re-establishes its inhibition of the laterals, again through the auxin mechanism (202a). It is not free auxin itself, however, but a specific inhibitor (131a) which is responsible for the absence of bud development during the dormant period.

In general the tall, rapidly growing single-shoot type of plant, which presumably produces and transports auxin efficiently, has few lateral branches, while shorter dwarf or stunted forms typically become bushy with numerous laterals or tillers. Auxin relations of this sort have been studied by van Overbeek (227,230) and Delisle (73) but much still remains to be done. Young leaves, since they are potent sources of auxin, exert a powerful inhibition (279) but mature leaves also inhibit in some plants (80). In guayule, a desert composite grown for its rubber content, the mature leaves actually inhibit the buds in their axils more powerfully than does the terminal bud (277). Indeed, a single leaf can inhibit the lateral buds all the way down the stem, a most unusual

behav-ior, which may well repay closer study. In Solidago plants in the rosette stage, each leaf inhibits somewhat the growth and development of the next, a phenomenon presumably parallel to that of bud inhibition

(101).

In the ferns, Albaum (1) has brought to light a parallel situation; the heart-shaped prothallia respond to the removal of their growing apex by formation of a new outgrowth (of the same shape as the indented area which they replace), and this "regeneration'' can be inhibited by apply-ing auxin in lanolin. Similarly, if the young sporophyte which develops later out of the prothallium is removed, another grows in its place, while application of auxin to the cut stump prevents this. These phenomena are thus quite parallel to the inhibition of buds, although buds as such are not involved. Doubtless Nature has provided many similar varia-tions on the same theme.

B. ROOT INHIBITION

Besides simple growth promotion, the first additional effect of auxin to be discovered was the inhibition of the elongation of roots. This was when Nielsen (217) extracted a crude auxin from cultures of the fungus Rhizopus suinus and showed that it promoted growth of the coleoptile but inhibited that of the root. The experiments were repeated and extended by Boy sen-Jensen (44) and Navez (214) and finally done with pure auxins by Kögl, Haagen Smit, and Erxleben. The technique is simply to immerse the roots of young seedlings in serial dilutions of the auxin and measure elongation with a millimeter scale. There is some thicken-ing, but this is not, as was first thought, sufficient to compensate for the decrease in length; the auxin therefore produces a large total decrease in root weight (312). The inhibition in length is roughly proportional to the logarithm of the concentration, so that the effect has been used as a simple auxin assay by Lane (184) and Bonner and Koepfli (34). Control of pH is essential (202), since auxin enters tissues much more readily in the free acid form than as an ionized salt (4,326). The activities of a number of substances have been compared in this way (92,184,202,311, especially 34) and it appears that, in general, compounds which have auxin activity as measured by growth promotion also cause root inhibi-tion; if inactive in the Avena or pea test they are inactive in inhibiting roots. The inactivity of indolecarboxylic, α,α-dimethyltoluic and trans-cinnamic acids (34) is of particular interest in connection with the relation between structure and activity discussed in Section III, C. Recently Thompson et al. (336) have published this as a new method, and tested 1060 compounds with it. Of these, the most active were: 2,4-dichloro-phenoxyacetic acid, its anhydride, sulfoanilide, and certain esters;

2-methyl-4-chlorophenoxyacetic acid, its anhydride, amide, some esters, and other derivatives ; 2-bromo-4-chlorophenoxyacetic acid; and 2-methyl-4-fluorophenoxyacetic acid. The first-mentioned is highly active in the curvature of slit pea stems (see Section II, D), though it gives only minute curvatures on Avena coleoptiles. Doubtless all these substances will be found to show growth-promoting activity on one or other of the standard growth-promoting auxin assays.

Of course, an inhibition is less specific than a growth promotion, and many compounds have some inhibiting effect in relatively high concen-trations. For this reason the inhibition of germination, studied by soak-ing seeds in solution and termed the "blastokolin" test (see Section A above) may not be very specific; it appears, however, to have no relation to the inhibition of root elongation by auxin. For instance, the ether-soluble growth inhibitor of tomatoes inhibits both root and shoot growth

(152). It is well known, too, that colchicine inhibits root elongation and causes characteristic swellings just proximal to the root tip (see, e.g., 82,192,201). It is perhaps remarkable that the changes in electric potential differences along the root which are caused by colchicine treat-ment are very similar to those caused by indoleacetic acid (338). This does not, of course, necessarily mean that, as Umrath and Weber (338) suggest, colchicine produces its effect by "activating" auxin in the root, for its effect on mitosis is far stronger than that of auxin. However, it is at least suggestive that the swellings induced by auxin in roots were shown by Levan (193) to contain many polyploid cells.

In contrast to the inhibition, extremely low auxin concentrations cause slight acceleration of root growth. This was discovered inde-pendently by a number of workers in 1936 (7,8,54,84,86,99,312); only Jost and Reiss (151) could find no acceleration. The effects are small but real; indoleacetic acid at 10~⁹M causes about 30% acceleration.

The response of roots to auxin is thus given by an optimum curve with its peak at excessively low auxin concentrations, as shown in Fig. 7.

Also, if the inhibition is not too great, it is accompanied by the formation of lateral-roots, i.e., by branching (151,312,372). The same effect results from decapitation of the root tip. However, such branching is not simply due to the inhibition of the tip growth, but is directly caused by auxin, because, as shown by Thimann (311), when auxin is applied to the base of the stem of Pisum, it slightly accelerates the growth rate of the main root, but still promotes the formation of laterals.

Short exposure of roots to auxin causes a temporary inhibition fol-lowed by a stimulation, which may lead to a general stimulation of growth of the entire plant (92,324). This "after-effect" is probably the cause

of the accelerated growth of "hormonized" seeds first reported by Cholodny with oats in 1936, and discussed further on p. 54. The dura-tion of the inhibidura-tion is propordura-tional to the time of exposure to the auxin, and Gast (92) has shown that the amount of stimulation which follows is roughly proportional to the amount of inhibition. A detailed analysis of the phenomena of root elongation will be found in the papers of Burström (54). He divides the process into a phase of increasing elastic-ity, which is accelerated by auxin, and one of decreasing elasticity (during which most of the growth takes place), which is inhibited by auxin.

AUXIN CONCENTRATION

FIG. 7.—Diagram of the inhibition and growth promotion of different organs as a function of auxin concentration. The abscissae for the bud and stem curves are only approximate. (From Thimann, 314.)

The effect of auxin in inhibiting root elongation acquired special interest as an explanation of the geotropism of roots (see 360, Chapter 9).

This geotropism, which is positive, i.e. toward gravity, would thus be due to the accumulation of auxin on the lower side as in shoots, but with the difference that the auxin would cause greater inhibition on the lower side.

This was the original Cholodny-Went theory of geotropism, but it has never been really rigidly established. While all experiments point in this direction, the closeness of the growing zone to the tip in many roots has made it extremely hard to obtain clear-cut growth responses after decapitation. The production of auxin by the root tip has also been hard to establish, in spite of many extraction and diffusion experiments (see especially 44,45,86,246,247,267). To sum up briefly many con-tradictory facts and interpretations (discussed by Fiedler, 86, and Thimann, 316), it appears clear now that small amounts of auxin are in fact regularly produced in the root tip provided it is adequately nourished (235,267). If this is to be enough so that its geotropic accumulation on

the lower side would account for positive curvature, it should also be enough to cause at least slight growth inhibition when the root is in the vertical position. In other words decapitation should cause slight acceleration of root growth. Some investigators have indeed found this effect, but agreement is not complete, perhaps due to the morphological difficulty mentioned above, which makes the length of the tip cut off extremely critical. It should be noted, too, that exposure to light increases the auxin content of isolated roots (267) and correspondingly inhibits elongation (253). Differences in lighting may thus also account for lack of agreement among different investigators.

Since high auxin concentrations also inhibit elongation of stems it might be supposed that stems supplied with considerably more auxin than they receive under physiological conditions should show positive, i.e., downward, geotropism. This has been claimed, indeed, by Geiger-Huber and Geiger-Huber (100) with mustard seedlings, but it is more probable that the downward curvature reported is not due to growth, but merely to plastic sagging, since Burkholder (53) has shown that similar

Since high auxin concentrations also inhibit elongation of stems it might be supposed that stems supplied with considerably more auxin than they receive under physiological conditions should show positive, i.e., downward, geotropism. This has been claimed, indeed, by Geiger-Huber and Geiger-Huber (100) with mustard seedlings, but it is more probable that the downward curvature reported is not due to growth, but merely to plastic sagging, since Burkholder (53) has shown that similar